1. Field Of The Invention
The invention relates to the identification, purification, and isolation of a novel protein which interacts with Bcl-2 protein to form heteromultimers (heterodimers) in vivo and more particularly to the purification, isolation and use of Bcl-2 associated protein, herein called Bax.
2. Description Of The Related Art
Cell death is an important aspect during the embryonic or post-natal development of major organ systems. Apoptosis, or programmed cell demise, also plays a critical role in maintaining homeostasis in many adult tissues. Within vertebrates; Bcl-2 is the best understood gene in a cell death pathway and functions as a cell death repressor.
Bcl-2 is unique among protooncogenes by being localized to the mitochondrial membrane as defined by Hockenbery, D. M., Nunez, G., Milliman, C., Schreiber, R. D. and Korsmeyer, S. J. xe2x80x9cBcl-2 is an inner mitochondrial membrane protein that blocks programmed. cell death.xe2x80x9d Nature 378, 334-336, 1990. Bcl-2 has been shown to have the oncogenic function of blocking programmed cell death whereas a deregulated Bcl-2 extends the survival of certain hematopoietic cell lines following growth factor deprivation. When pro-B-cell or promyelocyte cell lines are deprived of interleukin 3 they normally succumb to a programmed demise entitled apoptosis. This pattern of morphologic cell death is characterized by a dramatic plasma membrane blebbing, cell volume contraction, nuclear pyknosis, and internucleosomal DNA degradation following the activation of an endonuclease. Over expression of mitochondrial Bcl-2 appears to function as an antidote to this process and has the unique function of blocking programmed cell death independent of promoting proliferation.
The Bcl-2 protooncogene was discovered at the chromosomal breakpoint of the t(14;18) (q32;q21) which is the cytogenetic hallmark of human follicular lymphoma. Approximately 85% of follicular and 20% of diffuse B-cell lymphomas possess this translocation. Follicular lymphoma is often present as a low-grade malignancy composed of small resting IgM/IgD B cells. Over time, conversion to a more aggressive high-grade lymphoma with a diffuse large-cell architecture frequently occurs.
Studies of Bcl-2 emphasizes the existence of multiple pathways in the generation of neoplasia. The increased cell number in neoplastic tissue can be viewed as a violation of normal homeostasis. The maintenance of homeostasis in normal tissue, in many respects, reflects a simple balanced equation of input (cellular proliferation and renewal) versus output (cell death). This is most easily envisioned for encapsulated organs, such as the prostate, but is also true of the recirculating hematopoietic lineages. The maintenance of remarkably invariant cell numbers reflects tightly regulated death pathways as well as controlled proliferation. See for example S. J. Korsmeyer xe2x80x9cBcl-2 Initiates a New Category of Oncogenes: Regulators of Cell Deathxe2x80x9d, Blood Vol. 80 No. 4 pp. 879-886, Aug. 15, 1992.
Programmed cell death represents a cell autonomous suicide pathway that helps restrict cell numbers. The well-defined loss of specific cells is crucial during embryonic development as part of organogenesis. In mature tissues, genetically programmed demise regulates the volume of cells. A morphologically distinct and temporally regulated cell death entitled apoptosis has been identified by Wyllie A H: xe2x80x9cApoptosis; Cell death in tissue regulationxe2x80x9d. J. Pathol 153:313, 1987. Cells dying by apoptosis display marked plasma membrane blebbing, volume contraction, nuclear condensation, and the activation of an endonuclease that cleaves DNA into nucleosomal length fragments.
Bcl-2 has been localized to chromosome segment 18q21.3 in a telomere to centromere orientation. The Bcl-2 gene possesses 3 exons, the first of which is untranslated. Two potential promoter regions exist. P1 is GC rich with multiple SP1 sites and is used predominantly. Bcl-2 is an enormous gene in which a 225-kb intron II divides the protein encoding exons II and III. See Silvermann G A et al. xe2x80x9cMeiotic recombination between yeast artificial chromosomes yields a single clone containing the entire Bcl-2 proto-oncogenexe2x80x9d Proc Natl Acad Sci 87;9913, 1990. A molecular consequence of the translation is the movement of the Bcl-2 gene to the der(14) chromosome placing Bcl-2 in the same transcriptional orientation as the Ig heavy chain locus giving rise to chimeric RNAs. However, translocation does not interrupt the protein encoding region so that normal and translocated alleles produce the same sized, 25-Kd protein.
Hematopoietic progenitors, including pro-B cells, possess high levels of Bcl-2. See Hockenbery D, Zuter M, Hickey W, Nahm M, Korsmeyer S J: xe2x80x9cBcl-2 protein is topographically restricted in tissues characterized by apoptotic cell deathxe2x80x9d. Proc Natl Acad Sci USA 88:6961, 1991. Some mature B cells and, especially, B-cell lines have low levels of Bcl-2 RNA. In contrast, t(14;18)-bearing B cells have inappropriate elevated levels of the Bcl-2-Ig fusion RNA. Graninger W B, Seto M. Boutain B, Goldman, P, Korsmeyer S J: Expression of Bcl-2 and Bcl-2-Ig fusion transcripts in normal and neoplastic cells. J. Clin Invest 80:1512, 1987. This increased steady-state RNA reflects both increased transcription as well as a processing advantage for the Bcl-2-Ig fusion allele.
Bcl-2 has been introduced into a variety of interleukin (IL)-dependent cell lines to determine if it is involved in a growth factor pathway. See S J Korsmeyer above. Such lines were examined to determine if Bcl-2 would spare the need for a specific ligand/receptor interaction. However, no long-term growth factor-independent cell lines emerged after overexpression of Bcl-2 in IL-2, IL-3, IL-4, or IL-6 requiring lines. However, Bcl-2 conferred a death-sparing effect to certain hematopoietic cell lines after growth factor withdrawal in the IL-3-dependent early hematopoietic cell lines FDCP1, FL5.12, and 32D. This effect was not restricted to the IL-3/IL-3 receptor signal transduction pathway in that granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 deprived cells displayed a similar response. Yet, Bcl-2 enhanced cell survival was not universal, as neither IL-2-dependent T-cell lines nor an IL-6-dependent myeloma line showed a consistent effect upon factor withdrawal.
Bcl-2 has not been shown to directly promote cell cycle progression, nor does it necessarily alter the dose response to limiting concentrations of IL-3. See Nunez G, London L., Hockenbery D, Alexander M, McKearn J. Korsmeyer S J: xe2x80x9cDeregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell linesxe2x80x9d. J. Immunol 144;3602, 1990. Instead, Bcl-2 blocked the plasma membrane blebbing, volume contraction, nuclear condensation, and endonucleolytic cleavage of DNA known as apoptosis. Factor deprived cells return to Go, but do not die. However, they can be rescued after 30 days of deprivation by the addition of IL-3, indicating they are not terminally differentiated or permanently arrested.
While identifying the Bcl-2 cell death pathway is significant, a way of regulating the Bcl-2 pathway has not been discovered. The ability to down-regulate the effect of Bcl-2 would be advantageous in cancer therapy, in controlling hyperplasias such as benign prostatic hypertrophy (BPH) and eliminating self reactive clones in autoimmunity by favoring death effector molecules. Up-regulating the effect of Bcl-2 and favoring death repressor molecules would be beneficial in the treatment and diagnosis of immunodeficiency diseases, including AIDs, and in neurodegenerative and ischemic cell death.
The present invention relates to the unexpected discovery that Bcl-2 interacts with other proteins and in particular with an associated 21 kD protein called Bax. Bax shares extensive amino acid homology with Bcl-2 focused within highly conserved domains I and II. It has been unexpectedly discovered that Bax homodimerizes and forms heterodimers with Bcl-2 in vivo. It has also been discovered that overexpressed Bax accelerates apoptotic death induced by cytokine deprivation in an IL-3 dependent cell line and that overexpressed Bax also counters the death repressor activity of Bcl-2. This discovery provides a model in which the ratio of Bcl-2/Bax determines cell survival or death following an apoptotic stimulus.
Accordingly, one embodiment of the invention involves the formation of a purified and isolated Bcl-2 associated protein (Bax) and fragments thereof having the amino acid sequence of Domain I or II of FIG. 7.
Another embodiment involves the formation of Bcl-2 and Bax mutants wherein the native protein or fragment has at least one amino acid deleted or replaced by another amino acid and the mutants exhibits altered biological activity from the native protein or fragment.
Another embodiment involves an associated protein, which comprises Bax protein coupled with Bcl-2 associated protein or fragments thereof.
A further embodiment involves xe2x88x9d DNA isolate consisting essentially of a genomic DNA sequence encoding human Bax and more particularly a composition consisting of c DNA molecules which encode the Bax protein.
In one aspect of the invention, Bax polypeptides and compositions thereof are provided. Bax polypeptides comprise polypeptide sequences which are substantially identical to a sequence shown in FIGS. 3, 5 or 6. In one embodiment, the Bax polypeptide comprises domain I and/or domain II of the Bax polypeptide sequence, and preferably comprises the amino acid sequence(s) -W-G-R- and/or -Q-D-N-, and may be a cyclic polypeptide in some embodiments.
A further aspect involves a composition of xcex1RNA, xcex2RNA and/or xcex3RNA which encode a 21 kD, 24 kD or 4 kD Bax protein as well as cell lines producing such RNA species.
Another aspect of the invention involves Bax pharmaceutical compositions, which contain pharmaceutically effective amounts of a Bax polypeptide and a suitable pharmaceutical carrier.
Polynucleotide sequences encoding Bax are also provided. The characteristics of the cloned sequences are given, including the nucleotide and predicted amino acid sequence in FIGS. 3, 5 and 6. Polynucleotides comprising sequences encoding these amino acid sequences can serve as templates for the recombinant expression of quantities of Bax polypeptides, such as human Bax and murine Bax.
The invention also provides host cells expressing Bax polypeptides encoded by a polynucleotide other than a naturally-occurring Bax gene of the host cell.
In one aspect of the invention, a polynucleotide encoding a Bax polypeptide is delivered to a cell, such as an explanted lymphocyte, hematopoietic stem cell, bone marrow cell, and the like.
An additional aspect of the invention involves a method for controlling cell death repressor activity of Bcl-2, which comprises administering a Bax protein or fragment thereof to a cell containing Bcl-2 activity to enable the formation of a heterodimer containing Bax/Bcl-2, and inhibiting the Bcl-2 cell death repressor activity.
In one aspect of the invention, a method for modulating apoptosis of a cell, typically a lymphocyte, is provided. The method comprises administering to a cell an agent which alters intermolecular binding between Bcl-2 and Bax proteins, typically by inhibiting formation of heteromultimers (e.g., heterodimers) between Bcl-2 and Bax and/or homomultimers of Bcl-2 or Bax.
In one aspect of the invention, the method(s) of modulating apoptosis of a cell by administering an agent which alters intermolecular binding between Bcl-2 and Bax proteins are used to treat a pathological condition in a patient.
As an additional embodiment, the invention involves a method for assaying for the predisposition for an apoptotic cell death, which comprises: collecting a specimen to be tested; contacting the specimen with a material reactive with Bcl-2 or Bax protein; and detecting or determining the presence or absence of Bax and Bcl-2 protein and their ratio in the specimen.
The invention provides screening assays for identifying agents which modulate (e.g., inhibit) binding of a Bax polypeptide to a Bcl-2 polypeptide and/or which modulate (e.g., inhibit) binding of a Bax polypeptide to a Bax polypeptide.
The invention also involves the use of the protein Bax or Bcl-2 or mutant or fragment thereof for performing immunochemical methods for the detection and determination of the protein or its associated protein Bcl-2, in order to monitor cell survival versus death or to detect or monitor the course of diseases.
The invention also provides Bax polynucleotide probes for diagnosis of pathological conditions (e.g., neoplasia, AIDS, hyperplasia, congenital genetic diseases) by detection of Bax mRNA or rearrangements deletions or amplification of the Bax gene in cells explanted from a patient, or detection of a pathognomonic Bax allele (e.g., by RFLP or allele-specific PCR analysis).
In one aspect of the invention, transgenic nonhuman animals, such as mice, bearing a transgene encoding a Bax polypeptide and/or a Bcl-2 polypeptide are provided. Such transgenes may be homlogously recombined into the host chromosome or may be non-homlogously integrated.
Further included is a method for the treatment of a neurodegenerative disease, an immunodeficiency, or an ischemia, which comprises; increasing the effective amount of Bcl-2 or decreasing Bax or administering a mutant or fragment thereof to a patient to regulate the ratio of Bcl-2 to Bax to promote the survival of cells by generating an excess of Bcl-2; and, a method for the treatment of hyperplasias, hypertrophies, cancers and autoimmunity disorders, which comprises: decreasing the effective amount of Bcl-2 or increasing Bax or administering a mutant thereof to a patient to regulate the ratio of Bcl-2 to Bax so as to favor Bax and promote cell death.
In one aspect of the invention, an antisense polynucleotide is administered to inhibit transcription and/or translation of Bax in a cell.
The invention provides antibodies, both monoclonal antibodies and polyclonal antisera, which specifically bind to a Bax polypeptide with an affinity of about at least 1xc3x97107 Mxe2x88x921, typically at least 1xc3x97108 Mxe2x88x921 or more.